Antibacterial and herbicidal compounds and system for screening the same

ABSTRACT

A transgenic bacterial host cell that can be used as a screen for novel antibiotics and herbicides is provided. The genome of the transgenic bacterial host cell comprises disruptions in a first endogenous gene in the MEP pathway) and a transgene that functionally replaces the disrupted first gene. In other embodiments, the genome comprises a disruption in a first endogenous gene in the MEP pathway and a second endogenous gene which is located downstream of the first gene in the MEP pathway. A transgene that functionally replaces the disrupted downstream gene is cloned into the host cell. A mini operon containing the essential genes for the MVA pathway may also be cloned into the host cell. The transgenic host cell may be used in a method for screening compounds for antibiotic and herbicidal properties. The agents determined by the screening method may be used to kill bacteria or plants.

1. RELATED APPLICATIONS

This application is a 371 of PCT/US01/32143, filed Oct. 13, 2001, andthis application is related to and claims the benefit of U.S.Provisional Application No. 60/240,253 of C. Dale Poulter, filed Oct.13, 2000 and entitled “A Screening Novel Antibacterial and HerbicidalCompounds”, which is incorporated herein by this reference.

2. FIELD OF THE INVENTION

The present invention relates to antibacterial and herbicidal agents andsystems and methods of screening for antibacterial and herbicidalagents. More specifically, the invention relates to antibacterial andherbicidal agents that interfere with the methylerythritol phosphatebiosynthetic pathway for essential isoprenoid compounds and methods andsystems for screening the same.

3. TECHNICAL BACKGROUND

When penicillin first became widely available it was a medical miracle.With penicillin, doctors were able to treat disease that was previouslyuntreatable and often lethal. Soon, drug companies were mass-producingpenicillin and doctors were giving it to patients frequently.

However, shortly after the start of widespread use of penicillin,bacteria began to appear that were resistant to the antibiotic. Thefirst microbe to become resistant to penicillin was Staphylococcusaureaus. This bacterium is generally part of the harmless resident floraof the human body. However, it can also cause illness such as pneumonia.

Other bacteria began to show resistance to penicillin, and newantibiotics were discovered and introduced. With almost everyintroduction of a new antibiotic, bacteria have evolved a mechanism toresist the drug. Thus, bacteria have been found that are resistant tomethicillin, oxacillin, chloramphenicol, neomycin, terramycin,tetracycline, and cephalosporins. Many bacteria are resistant to anumber of types of antibiotics. Moreover, resistant bacteria cantransfer the resistance gene to other bacteria—even bacteria ofdissimilar species.

Despite the development of bacterial resistance to antibiotics,researchers have been able to discover new agents to battle the bugs.However, many of these new agents can also be toxic to humans. Moreover,the new agents may kill most of the resident microflora of thegastrointestinal tract, making the patient more susceptible to theinvasion of disease causing bacteria.

Vancomycin has been used as the drug of last resort in treatingmultiple-resistant bacterial strains. However, bacterial strains havebeen discovered that resist vancomycin, and many medical experts fearthat we will be without weapons to fight resistant bacteria.

Similar to the problems facing the treatment of bacterial disease arethose associated with herbicidal agents. Herbicidal agents have beenused by farmers, gardeners, and others for many years to combatundesirable plants and to increase crop yields. However, as withantibiotics, some plants have developed resistance to these compounds.Additionally, recent environmental regulations have caused some of themost effective herbicides to be discontinued because of potential harmto the environment and humans. Thus, there is a need to provide newherbicidal compounds that are less damaging to the environment and yeteffective at killing undesirable plants.

One potential target for finding compounds that are effectiveantibacterial and/or herbicidal agents is the pathways for the synthesisof isoprenoid compounds. Isoprenoid compounds form a large ubiquitousclass of natural products consisting of over 30,000 individual members.They fulfill a wide variety of cellular functions—such as components ofcell membranes (sterols), electron transport (ubiquinones), signaltransduction (prenylated proteins), photosynthetic pigments(chlorophylls), and cell wall biosynthesis (dolichols)—essential forviability.

Until recently, all isoprenoid compounds were thought to be synthesizedfrom acetyl-CoA by the widely accepted mevalonate (MVA) pathway found ineukaryotes and archaebacteria. Work stimulated by labeling patterns inbacterial hopanoids and ubiquinones inconsistent with their biosynthesisby the MVA pathway led to the discovery of a new independent route tothese molecules in many bacteria, green algae, and plants. In the newlydiscovered pathway, the five carbon atoms in the basic isoprenoid unitare derived from pyruvate and D-glyceraldehyde 3-phosphate (GAP).Pyruvate and GAP are condensed to give 1-deoxy-D-xylulose 5-phosphate(DXP).

In addition to serving as a precursor for the biosynthesis of thiamineand pyridoxol, DXP undergoes rearrangement and reduction to form2-methylerythritol 4-phosphate (MEP), the first committed intermediatein the MEP pathway for biosynthesis of isoprenoids. MEP is thencondensed with CMP to form a cytidine derivative, followed byphosphorylation of the C2 hydroxyl group and elimination of CMP to forma 2,4-cyclic diphosphate. The remaining steps to the fundamentalfive-carbon isoprenoid building blocks, isopentenyl diphosphate (IPP)and dimethylallyl diphosphate (DMAPP), have not been established. Formost organisms reported to date, the enzymes in the MEP pathway areencoded by essential single copy genes. The exception is a Streptomycesgram-positive strain, which contains both the MEP and MVA pathways.

DXP synthase (DXPase) lies just before the branch point to the Bvitamins and isoprenoids. Genes encoding the enzyme have been clonedfrom a number of species, including E. coli, Streptomyces sp. strainCL190, Mentha x piperiia, and Capsicum annuum. Disruption of the E. colidxs gene is lethal. DXPase catalyzes the decarboxylation of pyruvate andthe subsequent condensation of the thiamine bound two-carbonintermediate with GAP in a reaction similar to those catalyzed bytransketolases. Interestingly, DXPases contain regions with stronghomology to the E₁ subunit of pyruvate dehydrogenases and totransketolases. Although recombinant forms of the enzyme can use eitherGAP or D-glyceraldehyde as co-substrates with pyruvate, thephosphorylated form of the deoxy-sugar appears to be the normalintermediate in the pathway.

In light of the foregoing it would be a significant advancement in theart to provide a new method for screening for antibacterial andherbicidal agents. It would be a further advancement if the methodproduced compounds that were not toxic to humans and had decreasedeffect on the environment. An additional advancement would be to providecompounds that were effective antibiotics and herbicides. It would alsobe a significant advancement if an organism could be engineered for usein screening agents for activity against a large number of plants andbacteria. It would be a further advancement if the method could exploitthe MEP pathway which is present in plants and eubacteria. Suchorganisms, compounds, and methods are disclosed and claimed herein.

4. BRIEF SUMMARY OF THE INVENTION

The present invention provides a transgenic bacterial host cell whichcan be used to screen for novel antibacterial and herbicidal agents.Such a transgenic bacterial host cell may have a genome with adisruption in a first gene in the endogenous methylerythritol phosphate(MEP) pathway. The genome may also contain a disruption in the secondgene downstream from the first disrupted gene in the endogenous MEPpathway. Such disruptions may be an insertion or deletion that rendersthe endogenous gene functionally impaired. By functionally impaired, itis meant that the gene functions at a level below the normal wild-typefunction of the gene including non-functioning. The endogenous disruptedgene may be the DXP gene in the MEP pathway. However, other genes in theMEP pathway may be disrupted.

Most bacteria with a disruption in the MEP pathway are inviable. Thus,to culture such bacteria the disruption in the MEP pathway must berelieved. Such relief may be obtained by providing a chemical supplementthat bi-passes the disrupted gene in the pathway.

When the bacterial host cell has a disruption in only the first gene inthe MEP pathway, the genome of the transgenic bacterial host cell mayhave a cloned copy of a transgene that functionally replaces the firstgene. When the bacterial host cell has a disruption in a first andsecond gene in the MEP pathway, the genome of the transgenic bacterialhost may contain a cloned copy of a transgene that functionally replacesthe disrupted second gene. The transgene may be from the same species ofthe transgenic bacterial host or from another species allowing a testagent to be screened for specific activity against a particularorganism. The genome of the transgenic bacterial host cell may alsocontain a transgenic mevalonic acid (MVA) mini operon. The MVA operonprovides an alternate pathway to vital isoprenoid compounds.

Common laboratory bacterial strains may be used for the transgenicbacterial host. Such common bacterial strains may be E. coli or S.Typhimurium. E. coli and S. Typrimurium are easy to culture, readilyavailable, and do not pose a significant health threat to those in thelaboratory. It will be appreciated that other bacterial strains may beused as the host cell notwithstanding their easy of culture,availability, and virulence.

The invention also provides a method of screening, novel antibacterialand herbicidal agents. The method can include the step of obtaining atransgenic bacterial host cell. The transgenic bacterial host cell has agenome as described above with a disruption in a first or a first andsecond gene in the MEP pathway and a cloned copy of a transgene thatfunctionally replaces the disrupted first or second gene.

The transgenic bacterial host cell is cultured in a test culture. Themedia of the test culture contains a chemical supplement to relieve theblock in the first gene. The test culture is contacted with a testagent. A control culture of the transgenic bacterial host cell is alsogrown in media containing a chemical supplement to relieve the block inthe first gene. The control culture is not contacted with the testagent.

After a period of time the growth of the transgenic bacterial host cellsin the test and the control cultures is observed and compared. When anagent renders the transgenic bacterial host cell inviable as compared tothe control culture, then the test agent is a potential antibacterial orherbicidal agent.

When the transgenic bacterial host cell also comprises a MVA minioperon, the screening method may contain an additional control culturethat pinpoints the mode of action of the test agent to the MEP pathway.The screening method thus includes the step of growing the test cultureon media that contains a chemical supplement to relieve the block of thefirst disrupted gene in the MEP pathway. A control culture is grown inmedia containing MVA. Both the control culture and the test culture arecontacted with a test agent. After a period of time the test culture andthe control culture are observed and the growth of the transgenic hostobserved. A test agent that renders the test culture inviable and doesnot affect the viability of the control culture may be an effectiveantibacterial or herbicidal agent.

When the MVA mini operon is present in a bacterial host cell containinga disruption in the first gene in the MEP pathway and a cloned copy of atransgene that functionally replaces the first gene, the bacterial hostcell will grow on minimal medium. The screening method may includecontacting the transgenic host cell growing on minimal media with a testagent. A control can be made by contacting the transgenic host cellgrowing on media supplemented with MVA. When the test agent renders thetransgenic host cell growing on minimal medial inviable and does notaffect the viability of the culture on the media supplemented with MVA.The agent maybe an effective inhibitor of the cloned gene in the MVApathway and thus an effective antibacterial or herbicidal agent.

The transgene that functionally replaces the first or second disruptedgene in the transgenic host cell may be from the same species as thehost cell or from another species. When a transgene is used from aspecies other that the host cell, an agent may be tested for disruptingthe gene of the specific organism from which the transgene originated.Thus, the test agent may be tested for its ability to kill the specificorganism.

A method of selectively killing an organism such as bacterial cell or aplant is also provided. The method includes the step of contacting theorganism with an antibacterial agent or herbicidal agent other thanfosmidomycin which selectively inhibits the methylerythritol phosphate(MEP) pathway of the bacterial cell. The ability of the agent toselectively inhibit the MEP pathway of the organism cell can bedetermined by the screening method of the present invention. Thus, atransgenic bacterial host cell is obtained whose genome comprises adisruption of a first endogenous gene or a first and a second endogenousgene in the MEP pathway. The first gene may be a disruption in theendogenous DXP gene. The genome also contains a transgene obtained froman organism that functionally replaces the disrupted first or secondgene. The transgenic host cell is then cultured on media containing achemical supplement to relive the disruption of the first gene. A testculture is contacted with a test agent. The growth of the transgenichost cell in the presence of the test agent is compared to a controlculture. An agent that inhibits the growth of the test culture indicatesthat the agent may inhibit the MEP pathway of the organism.

In order to determine if the mode of activity of the agent is theselective inhibition of the MEP pathway the bacterial host cell may alsocomprise a MVA mini operon. The activity of the test agent is determinedby the step of growing the test culture on media that contains achemical supplement to relieve the block of the first disrupted gene inthe MEP pathway. A control culture is grown in media containing MVA.Both the control culture and the test culture are contacted with a testagent. After a period of time the test culture and the control cultureare observed and the growth of the transgenic host observed. A testagent that renders the test culture inviable and does not affect theviability of the control culture may be an effective antibacterial orherbicidal agent.

5. SUMMARY OF THE DRAWINGS

A more particular description of the invention briefly described abovewill be rendered by reference to the appended drawings and graphs. Thesedrawings and graphs only provide information concerning typicalembodiments of the invention and are not therefore to be consideredlimiting of its scope.

FIG. 1 is a schematic representation of the Methylerythritol Phosphate(MEP) Pathway.

FIGS. 2A-2F are photographs showing the E. coli FH11 at 30° C. and 40°C.

6. DETAILED DESCRIPTION OF THE INVENTION

Isoprenoid compounds represent one of the largest and most diversegroups of natural products, with over 30,000 identified members to date.Poulter, C. D., & Rilling, H. C. In Biosynthesis of IsoprenoidCompounds; Porter, J. W., & Spurgeon, S. L., Eds., Wiley: New York,1981; Vol. 1, pp. 161-224. These molecules perform a variety ofimportant tasks in their host organisms, including serving as hormonesin mammals, antioxidants in plants, and electron carriers duringcellular respiration. Sacchetini, J. C., & Poulter, C. D. Science 277:1788-1790 (1997). Isopentenyl diphosphate (IPP)¹ and dimethylallyldiphosphate (DMAPP) are the five-carbon building blocks used toconstruct more complicated isoprenoid structures.

Until recently, IPP and DMAPP were thought to originate from acetate viathe mevalonate pathway. Bochar, D. A., et al. In Comprehensive NaturalProducts Chemistry; Barton, D., & Nakanishi, K., Eds., Elsevier: Oxford,1999; Vol. 2, pp. 15-44. However, studies by Rohmer and Arigoniuncovered an alternate pathway that operates in plants, algae, andbacteria where IPP and DMAPP are derived from pyruvate andglyceraldehyde-3-phosphate. Flesch, G., & Rohmer, M., Eur. J. Biochem.175: 405-411 (1988); Arigoni, D., et al., Proc. Natl. Acad. Sci. U.S.A.,94: 10600-10605 (1997); Eisenreich, W., et al. Chem. Biol., 5: R221-R233(1998). Referring to FIG. 1, the two three-carbon precursors are joinedin a thiamine diphosphate-mediated condensation catalyzed by 1-deoxy-D-xylulose-5-phosphate (DXP) synthase to give DXP. Lois, L. M.,et al., Proc. Natl. Acad. Sci. U.S.A., 95: 2105-2110 (1998). DXP is thenrearranged and reduced by 2-C-methyl-D-erythritol-4-phosphate synthase(MEP synthase) (also called DXP reductoisomerase or DXPisomeroreductase) to form MEP. DXP, as well as its dephosphorylatedcounterpart deoxyxylulose, have also been identified as intermediates inthe synthesis of vitamins B₁ and B₆. Begley, T. P., Nat. Prod. Rep., 13:177-185 (1996); Cane, D. E., et al., J. Am. Chem. Soc., 121: 7722-7723(1999). Thus, MEP is the first intermediate committed to IPP formation,and the name “methylerythritol phosphate pathway” has recently beensuggested for this route (4^(th) European Symposium on PlantIsoprenoids, Barcelona 1999). MEP is converted to2-C-methyl-D-erythritol-2,4-cyclodiphosphate by the action of threesuccessive enzymes.Rohdich, F., et al., Proc. Natl. Acad. Sci. U.S.A.,96: 11758-11763 (1999); Kuzuyama, T., et al. Tetrahedron Lett., 41:703-706 (2000); Luttgen, H., et al., Proc. Natl. Acad. Sci. U.S.A., 97:1062-1067 (2000); Kuzuyama, T., et al. Tetrahedron Lett., 41: 2925-2928(2000); Herz, S., et al., Proc. Natl. Acad. Sci. U.S.A., 97: 2426-2490(2000); Takagi, M., et al.Tetrahedron Lett., 41: 3395-3398 (2000).

Many bacteria, including the pathogens involved in human disease, manyof which are becoming drug-resistant, rely on the recently discoveredmethylerythritol phosphate (MEP) pathway for the biosynthesis ofessential isoprenoid compounds. Deletion of genes in the pathway ordisruption on enzyme activity by inhibitors is lethal.

E. coli strains with disruptions in the genes encoding enzymes in MEPpathway are not viable. This is the only pathway for biosynthesis ofessential isoprenoid compounds in E. coli and many other bacteria. TheMEP pathway is also found in plant chloroplasts and in some speciesmaybe required for biosynthesis of chlorophyll and photoprotectivecarotenoids. Gene disruptions in the MEP pathway can be complemented (1)chemically by supplementation with chemical intermediates in thepathway, (2) by plasmid encoded copies of the disrupted gene, and (3) bya mini-operon that encodes enzymes that synthesize isopentenyldiphosphate from mevalonate (MVA) and, thus, by-passes the MEP pathway.

Bacterial strains such as E. coli and S. typhimurium containing (1) adisruption in a first gene in the MEP pathway such as the wild-type genefor deoxyxylulose phosphate (DXP) and a disruption in a second genedownstream from the first gene in the MEP pathway, (2) a cloned-in copyof the downstream gene from the host or another organism, and (3) a MVAmini-operon are inviable when grown on rich or minimal media, but areviable when the medium contains a chemical supplement to relieve theblock of the first gene or MVA. Addition of a compound to the mediumthat inhibits the plasmid-encoded downstream gene renders the straininviable on media containing the chemical supplement but viable onMVA-supplemented media.

Likewise bacterial strains containing (1) a disruption in a first genein the MEP pathway such as the wild-type gene for deoxylulose phosphate(DXP) (2) a cloned-in copy of the disrupted first gene from the host oranother organism, and (3) a MVA mini-operon are inviable when grown onrich or minimal media, but are viable when the medium contains achemical supplement to relieve the block of the first gene or MVA.Addition of a compound to the medium that inhibits the plasmid-encodeddownstream gene renders the strain inviable on media containing thechemical supplement but viable on MVA-supplemented media.

These strains can be used to screen for inhibitors of these enzymesencoded by the downstream-disrupted genes by comparing viability in thepresence and absence of chemical supplements relative to MVA. This is ahighly selective method to screen for molecules that inhibit isoprenoidbiosynthesis by targeting enzymes in the MEP pathway. The screen isselective for the cloned enzyme in an easily manipulated host,regardless of its source. This permits one to screen for inhibitors ofenzymes from pathogens in a safe background and eliminates much of thecost and danger of discovering lead compounds effective againstpathogens.

The MEP pathway is only found in eubacteria and plant chloroplasts.Other eukaryotic organisms and archaebacteria synthesize isoprenoidcompounds by the MVA pathway and are not subject to the toxicityassociated with inhibitors that are highly selective for enzymes in theMEP pathway.

The present invention provides a transgenic bacterial host cell whichcan be used to screen for novel antibacterial and herbicidal compounds.The genome of the transgenic bacterial host cell has disruption a in allendogenous gene of the MEP pathway. In certain embodiments, the gene isthe dxs gene, the disruption of which blocks the DXP pathway of thebacterial host cell. An additional disruption of a second endogenousgene which is located downstream in the MEP pathway is also present. Agene which functionally replaces the disrupted downstream gene is clonedinto the host cell from an organism such as a plant or bacterium. Incertain embodiments, an MVA mini operon may be cloned into the hostcell.

As used herein, the term “genome” refers to all of the genetic elementsin a cell, including without limitation the genes located on chromosomesand extrachromosomal elements (such as plasmids).

It may be desirable that bacterial strains that can readily and safelybe grown in the laboratory be used as the host cell. Such bacterialstrains include E. coli and S. typhimurium.

The transgenic bacterial host cells may be used in a method of screeningfor antibacterial and herbicidal agents. The transgenic cells arecultured in media containing a chemical supplement that lifts the DXPblock. The transgenic host cells are then contacted with a test agent.The cell growth of the test culture is compared to the growth in acontrol culture. Inviability of the host cell in the test culturecompared to viability in the control culture indicates that the agent iseffective of selectively killing the organism from which the transgenewas obtained.

In certain embodiments in which the transgenic host cell contains acloned-in MVA mini operon, the control culture may be a transgenic hostcell grown in the presence of MVA, the chemical supplement that liftsthe DXP block, and the test agent. When the transgenic hosts cells areinviable in the test culture and viable in the control culture, the testagent is an effective antibacterial or herbicidal agent against theorganism.

The invention also provides methods of killing plants and bacteria withantibacterial and herbicidal agents determined by the methods of thepresent invention. Because these agents work via the MEP pathway, whichis not found in eukaryolic cells other than in plant chloroplasts, theseagents exhibit minimal toxicity and adverse effect on the environment.The method of selectively killing plants and bacteria uses an agent thatselectively inhibits the MEP pathway of the plant or bacteria. Theability of an agent to selectively inhibit the MEP pathway of theorganism can be determined by the screening methods of the invention. Anagent that is determined to selectively inhibit the MEP pathway of anorganism can be used to kill the organism by contacting the organismwith the agent.

All publications, patents, patent applications, and commercial materialscited in this application, including those cited in the Appendix, arehereby incorporated by reference in their entirety.

In some embodiments, a transgenic bacterial host cell comprising agenome is disclosed. This genome may comprise a disruption of a firstgene, wherein the first gene is an endogenous gene in themethylerythritol phosphate (MEP) pathway and a transgene thatfunctionally replaces the disrupted first gene. In some embodiments, thegenome may further comprise a transgenic mevalomc acid (MVA) minioperon. In other embodiments, the first gene is an endogenous dxs gene.In other embodiments the bacterial host cell is selected from the groupconsisting of E. coli and S. Typhimurium. In further embodiments, thetransgene is from a species other than the bacterial host cell. Otherembodiments have the transgene being from the same species as thebacterial host cell.

In other embodiments, a transgenic bacterial host cell comprising agenome is disclosed. The genome comprises a disruption of a first gene,wherein the first gene is an endogenous gene in the Methylerythritolphosphate (MEP) pathway, a disruption of a second gene, wherein thesecond gene is an endogenous gene downstream from the first gene in theMEP pathway, and a transgene that functionally replaces the disruptedsecond gene.

Embodiments also relate to a method of screening antibacterial andherbicidal agents for activity against an organism, the methodcomprising the step of obtaining a transgenic bacterial host cell whosegenome comprises a disruption of a first gene, wherein the first gene isan endogenous gene in the Methylerythritol phosphate (MEP) pathway, adisruption of a second gene, wherein the second gene is an endogenousgene downstream from the first gene in the MEP pathway, and a transgeneobtained from the organism that functionally replaces the disruptedsecond gene. The step of culturing a test culture of the transgenicbacterial host cell in media containing a chemical supplement to relievethe disruption of the first gene is also added. An additional step ofcontacting the transgenic bacterial host cell of the test culture with atest agent may be used. The step of culturing a control culture of thetransgenic bacterial host cell in media containing a chemical supplementto relieve the disruption of the first gene may be used. Also, the stepof comparing growth of the bacterial host cell contacted by the testagent to the growth of the bacterial host cell of the control culture,wherein the killing of the transgenic bacterial host cell of the testculture and the viability of the transgenic bacterial host cell of thecontrol culture indicates that the test agent is an effectiveantibacterial or herbicidal agent against the organism may be used inthe method.

Further embodiments may be designed in which the genome furthercomprises a transgenic mevalonic acid (MVA) mini operon, and the methodfurther comprises growing a control culture of the bacterial host cellon media containing MVA, such that when an agent renders the bacterialhost cell inviable in media containing a chemical supplement to relivethe disruption of the first gene and viable on media containing MVA, theagent is an effective antibacterial or herbicidal agent against theorganism.

In other embodiments, a method of screening antibacterial and herbicidalagents for activity against an organism is disclosed. The methodcomprises the step of obtaining a transgenic bacterial host cell whosegenome comprises a disruption of a first gene, wherein the first gene isan endogenous gene in the Methylerythritol phosphate (MEP) pathway. Thestep of culturing a test culture of the transgenic bacterial host cellin media containing a chemical supplement to relieve the disruption ofthe first gene may also be added. The step of contacting the transgenicbacterial host cell of the test culture with a test agent may also beadded. The step of culturing a control culture of the transgenicbacterial host cell in media containing a chemical supplement to relievethe disruption of the first gene. The step of comparing growth of thebacterial host cell contacted by the test agent to the growth of thebacterial host cell of the control culture may further be added. Thekilling of the transgenic bacterial host cell of the test culture andthe viability of the transgenic bacterial host cell of the controlculture indicates that the test agent is an effective antibacterial orherbicidal agent against the organism. In additional embodiments, themethod is designed in which the genome further comprises a transgeneobtained from the organism that functionally replaces the disruptedfirst gene, and wherein the test culture of the transgenic bacterialhost cell is cultured in media containing a chemical supplement toactivate the transgene and the control culture of the transgenicbacterial host cell is cultured in media containing a chemicalsupplement to activate the transgene.

4. EXAMPLES

The following examples are given to illustrate various embodiments whichhave been made with the present invention. It is to be understood thatthe following examples are not comprehensive or exhaustive of the manytypes of embodiments which can be prepared in accordance with thepresent invention.

Example 1 Construction of an E. Coli Strain Containing an Insert in theDeoxyxylulose Synthase Gene that Contains the Genes for ConvertingMevalonic Acid to Isopentenyl Diphosphate

An E. coli strain was constructed with a disruption in the gene forsynthesis of deoxyxylulose (dxs), where the disruption consisted of anoperon that coded for the yeast enzymes necessary to convert mevalonicacid (MVA) to isopentenyl diphosphate (IPP) under control of anarabinose promoter. This strain is capable of growing on mediasupplemented with methylerythritol (ME) by restoring isoprenoidmetabolism through the methylerythritol phosphate (MEP) pathway or whensupplemented with MVA through the MVA pathway.

Cloning E. coli dxs: The E. coli deoxyxylulose synthase gene (dxs) wasPCR amplified using the following primers containing BamHI (bold) andKpnI (italics) restriction sites:5′-GGTACCATGAGTTTTGATATTGCCAAATACCCG-3′ (SEQ ID NO: 1) (dxsPCR1) and5′-GGATCCTTATGCCAGCCAGCCAGGCC-3′ (SEQ ID NO: 2). The PCR products wereligated directly into the pGEM-T Easy vector (Promega) to give pGEM-dxs.Upon restriction with BamHI and KpnI, fragment containing dxs was clonedinto the BamHI/KpnI site of pBluescript SK+ (Stratagene) to givepBS-dxs.

Construction of pCAT53-3: A cassette containing the kanamycin resistancegene was obtained by restriction of plasmid pUC4K (Pharmacia) with SalI,cloning the fragments into the XhoI restriction site of pFCO1 to givepFCO1-kan containing the yeast genes for mevalonate kinase (erg8),mevalonate diphosphate decarboxylase (erg12) and phosphomevalonagekinase (erg19). The araC gene and the P_(BAD) promotor were obtained byrestriction of pBAD-gIII A (Invitrogen) with NsiI, filling in the endswith Klenow, and restriction with SacI. The gene cluster was cloned intopFCO1-kan that had been digested with SapI and filled in with Klenow andthen restricted with SacI to give pCAT53-3.

Disruption of dxs with the mevalonate cluster: pCAT53-3 was restrictedwith PspOMI, the sticky ends were filled in with Klenow, the DNA wasrestricted with BsrBI, and the araC/P_(BAD) DNA was cloned into HindIIIrestricted pBS-dxs using Chain Reaction Cloning to formpBS-dxs::pCAT53-3. E. coli strain JC7623 was transformed with plasmidpTP223. Poteete, A. R., et al., Virology 134: 161-167 (1984). To enhancehomologous recombination. PBS-dxs::pCAT53-3 was linearized byrestriction with SpeI and BsrBI and used to transform JC7623/pTP223 byelectroporation. The transformants were plated on LB containing 40 μg/mLkanamycin and 50 μg/mL methylerythritol (ME). After two days at 37° C.,the colonies were replicate plated on LB/kan/ME and LB/kan. Thosecolonies that grew on LB/kan/ME but not on LB/kan were plated onLB/kan/ME, LB/kan, and LB/kan/sodium mevalonate (5 mM)/0.02% arabinose(ara). Colonies that grew on LB/kan/ME and LB/kan/MVA/ara but not LB/kanwere stored as glycerol stabs and frozen DMSO stocks.

Example 2 Construction of a S. Typhimurium Strain Containing an Insertin the Deoxyxylulose Synthase Gene that Contains the Genes forConverting Mevalonic Acid to Isopentenyl Diphosphate

A S. typhimurium strain was constructed with an insertion in the genethat codes deoxyxylulose synthase (dxs). This insertion consisted of anoperon, under the control of an arabinose promoter, which included theyeast genes necessary to convert mevalonic acid (MVA) to isopentenyldiphosphate (IPP). This strain is viable when supplemented withmethylerythritol (ME) or MVA and arabinose, which restore inbiosynthesis of IPP through the methylerythritol phosphate (MEP) pathwayor MVA pathway respectively.

Cloning of S. typhimurium dxs: The S. typhimurium dxs gene was PCRamplified using primers sDXS1 and as DXS1. Primer sDXS1 is homologous tothe upstream gene ispA while asDXS1 displayed homology to the regionbetween dxs and the downstream gene yajO. The 2 kb PCR product waspurified using QIAquick PCR purification kit (Qiagen) and then ligatedinto the vector pGEM-T (Promega) as directed by the manufacturer to givepRMC13. The ligation reactions were used to directly transform DH5αsub-cloning efficiency cells (Gibco-BRL) following the manufacturersinstruction. The construct was verified by small scale plasmidpurification using Qiagen's QIAprep spin miniprep kit followed byrestriction digest with SacII.

Construction of pCAT53.3: A cassette containing the kanamycin resistancegene (Kan^(R)) was obtained by restriction of plasmid pUC4K (Pharmacia)with SaII. The Kan^(R) fragment was then ligated into pFCO1, a plasmidwhich contains the yeast genes for mevalonate kinase (erg8), mevalonatediphosphate decarboxylase (erg12) and phosphomevalonate kinase (erg19),which had been digested with XhoI to produce pFCO1-kan. Hahn, F. M. etal. J. Bacteriol 183: 1-11 (2001) A cassette containing the gene araCand the P_(BAD) promoter was obtained by first digesting pBAD-gIIIA(Invitrogen) with NsiI, then blunt ending by treatment Klenow, andfinally restricting with SacI. This araC/P_(BAD) cassette wasdirectionally ligated into the SacI site and Klenow treated SapI site ofpFCO1-kan resulting in plasmid pCAT53.3.

Construction of pRMC14: Plasmid pRMC13 was digested with EcoRV andBsu361 to remove ˜1000 bases from the middle of the dxs gene. PlasmidpCAT53.3 was digested with BstBI and PspOMI to liberate a fragmentcontaining araC, PBAD, erg8, erg12, erg19 and Kan^(R) (MevCluster)Fragments Bsu361-pRMC13-EcoRV and BsrBI-MevCluster-PspOMI were filled inwith Klenow and directionally ligated using chain reaction cloning (CRC)with the primers crcKANDXS and crcDXSARA to form pRMC14. Pachuk, C. J.et al., Gene 243: 19-25 (2000). The CRC reactions were used to transformDH5a max-efficiency cells (Gibco-BRL) according to manufacturersinstructions. The plasmid construct was verified by small scale plasmidpurification using Qiagen's QIAprep spin miniprep kit followed byrestriction digest with HindIII.

Disruption of dxs with the mevalonate cluster: Plasmid pRMC14 waselectroporated into S. typhimurium strain TR6579 (obtained from JohnRoth, University of Utah) to produce strain RMC24. A medium scaleplasmid purification was performed on this strain using Qiagen-tip 100columns. Plasmid pRMC14, from this preparation, was digested with ApaIand SpeI to liberate a linear fragment containing the MevCluster, araC,P_(BAD), and flanking regions of dxs homology. This linear fragment waselectroporated into S. typhimurium strain TT22236 (LT2 harboring plasmidpTP223 obtained from John Roth, University of Utah) which contains aplasmid copy of the λ-Red genes to enhance homologous recombination.Poteete, A. R. & Fenton, A. C., Virology 134: 161-167 (1984).Recombinants were selected for on LB media containing Kan (50 μg/mL) andME (50 μg/mL). A strain with insertion into dxs (RMC25) was isolated byreplica plating to media with and without ME and screening fortransformants viable only in the presence of ME. Insertion into dxs wasverified by amplifying the regions of insertion (from inside the insertinto the surrounding regions) using PCR with primers sISPA1 with asARAC1and sKAN1 with asYAJO1 followed by sequencing. Strain RMC25 was thenused as a donor to prepare a standard P22 HT105/λ int-201 lysate whichwas used to transduce dxs::pCAT53.3 into S. typhimurium strain LT2resulting in strain RMC26 (S. typhimurium LT2 dxs::pCAT53.3). Achmieger,H., Mol. Gen. Genet. 110, 378-381 (1971). Again, this construct wasverified by phenotype, as well as, PCR and sequencing.

TABLE 1 Primer Sequences Primer Sequence sDXS15′-CTGATAGAGGACGCCCGTCA-3′ (SEQ ID NO: 3) asDXS15′-CATAGCAGGAGCAAAGAGGG-3′ (SEQ ID NO: 4) crcKANDXS5′-TCGAGGTCGAGGGGGGGCCTGAG (SEQ ID NO: 5) GGGCGAAAGTCGTAAA-3′ crcDXSARA5′-CAATGAATCACGCAGGCGATCTC (SEQ ID NO: 6) GCCGCAGCCGAACGACC-3′ sISPA15′-CGTTAGACTTGGGCGTTGAG-3′ (SEQ ID NO: 7) asARAC15′-CTTTGAGCACCACCCGGAT-3′ (SEQ ID NO: 8) sKAN15′-GGCAGAGCATTACGCTGACT-3′ (SEQ ID NO: 9) asYAJO15′-AGTTAATGCCGCCCTCAAGG-3′ (SEQ ID NO: 10)

TABLE 2 Genotype of Strains of S. typhimurium LT2 and E. coli K-12Strain Genotype Plasmid Ref./Source LT2^(a) Wild type LB5010^(a) MetA22MetE551 trpD2 Bullas, L. R. ilv-452-leu-pro- & Ryu, J. (leaky) hsdLT6 JBacteriol hsdSA29 hsdB-strA 120 156:471-474 galE- (1983) TT22236^(a)Wild type TP223 RMC24^(a) MetA22 MetE551 trpD2 pRMC14 N/Ailv-452-leu-pro- (leaky) hsdLT6 hsdSA29 hsdB-strA 120 galE- RMC25^(a)dxs520::MevOperon N/A Δdxs520-1452 RMC26^(a) dxs520::MevOperon N/AΔdxs520-1452 DH5α^(b) F-F80dlacZDM15 D Gibco-BRL (lacZYA-argF)U169 deoRrecA1 endA1 phoA hsdR17(rK-.mK-) supE44 I-thi-1 gyrA96 reIA1 ^(a)Strainsof S. typhimurium LT2 ^(b)Strains of E. coli K-12

Example 3 Screen for Antimicrobial Agent Using Strain JC7623dxs::pCAT53-3

Strain JC7623 dxs::pCAT53-3 was made chemically competent, transformedwith pLME10 (R. capsulatus dxsB in pET11-A, manuscript in press) andgrown in LB/kan/IPTG (kan^(r) is in disruption and IPTG is for inductionof plasmid derived dxs). A 5 μL portion of a 1,000 fold dilution wasplated on plates as follows.

Plate a contained LB/kan/IPTG. Plate B contained LB/kan. Plate Ccontained LB/kan/MVA/arabinose. Plate D contained LB/kan/ME. Plate Econtained LB/ka/IPTG/fosmidomycin. Plate F containedLB/kan/MVA/fosmidomycin. Plate G contained LB/kan/ME/fosmidomycin. Thesupplements in the media were present at the following concentrations:kan (40 mg/L), IPTG (1 mM), MVA (5 mM), arabinose (0.02%), ME (50 mg/L),fosmidomycin (˜4 μg/mL).

After a period of 1 to 2.5 days the plates exhibited the followinggrowth. Plate A slowed microcolonies after 1.5 days. Plate B showedmicrocolonies after 1.5 days. Plate C showed microcolonies after 1 day.Plate D showed colonies after 1 day. Plate E showed no growth after 2.5days. Plate F showed microcolonies after 1 day. Plate G showed coloniesafter 1 day.

The growth rate of the plates was compared after 2.5 days. The growth onthe plates D and G were equal. The growth on plates A and B were equaland less than the grown on plates D and G. The growth on plates C and Fwere equal and less than the growth on plates A and B. Plate E exhibitedno growth.

These results demonstrate (1) that the disruption of dxs in themethylerytheilol phosphate (MEP) pathway for isoprenoid biosynthesis inE. coli can be complemented by a plasmid-encoded copy of the same enzymefrom a different organism, (2) that a known inhibitor of the MEP pathwayblocks growth, and (3) that growth is restored by supplementation withmevalonate (MVA) is provided along with the inhibitor. Thus, inhibitorsof the MEP pathway block growth, and restriction of growth by MVAprovides a positive control that pinpoints the mode of action of theinhibitor to the MEP pathway.

Example 4 Screen for Antimicrobial Agent Using Strain RMC25

Strain RMC25 was made electrocompetent, electroporated with pLME10 (R.capsulatus dxsB in pET11-A), and grown in LB/kan/amp/ME (kan^(r) is inthe DXS disruption and amp^(r) is in pET11-A). A 5 μL portion of a 1,000fold dilution was plated on plates A through G. Each plate containedsupplements as follows. Plate A contained LB/kan/IPTG. Plate B containedLB/kan. Place C contained LB/kan/MVA/arabinose. Plate D containedLB/kan/ME. Plate E contained LB/kan/IPTG/fosmidomycin. Plate F containedLB/kan/MVA/fosmidomycin. Plate G contained LB/kan/ME/fosmidomycin.Concentrations for the supplements are as follows: kan (40 mg/L), IPTG(1 mM), MVA (5 mM), arabinose (0.02%), ME (50 mg/L), fosmidomycin (˜4μg/mL).

After 1 to 2.5 days growth was observed on the plates as follows. PlateA showed colonies after 1.5 days. Plate B showed microcolonies after 1.5days. Plate C showed colonies after 1 day. Plate D showed colonies after1 day. Plate E showed no growth after 2.5 days. Plate F showed coloniesafter 1 day. Plate G showed colonies after 1 day.

The growth rate of the plates was compared after 2.5 days. The growth onthe plates D and G were equal. The growth on plates A was less than thegrown on plates D and G. The growth on plates C and F were equal andless than the growth on plate A. The growth on plate B was less than thegrowth on plates C and F. Plate E exhibited no growth.

These results are demonstrate a constructed S. typhimurium strain can beused as a screen for drugs targeting the MEP pathway. The disruptedstrain has the chromosomal copy of DXS disabled. This is a lethalcondition when the strain is grown on LB media that does not containmevalonate. The DXS disruption is complemented with a plasmid-encodedcopy of one of the genes for DXS from R. capsulatus. The complementedstrain grows on LB with a wt phenotype. When fosmidomycin, a compoundthat inhibits MEP synthase, is added to the media, growth is inhibited.Growth is restored when mevalonate is added to the media. Thus, theability to screen for inhibition of a plasmid-encoded heterologousenzyme in the MEP pathway by monitoring growth of RMC25/pLME10 isdemonstrated. Extensions of this procedure to other enzymes in the MEPpathway from other organisms are obvious and straightforward.

Example 5 Synthesis of IPP from MVA in E. Coli

The three yeast genes ERG8, ERG12, and ERG19 were isolated by PCR andwere translationally coupled with overlapping start and stop codons inthe synthetic operon contained within pFCO1. The PCR primers for allthree yeast genes contained additional nucleotides for theribosome-binding site AGGAGGAG (SEQ ID NO: 12) and the insertion ofGln-Glu-Glu-Phe (SEQ ID NO: 11) at the C-terminus of the encodedproteins to facilitate their purification using a monoclonal antibodycolumn. The gene for mevalonate idnase, ERG12, served as the foundationfor the construction of the operon. ERGS, the gene for phosphomevalonatekinase, was added upstream of ERG12, and ERG19, the gene for mevalonatediphosphate decarboxylase, was appended to the 3′ end of ERG12. In orderto ensure specific annealing to ERG12, the additional nucleotidescorresponding to codons for the N-terminus of mevalonate diphosphatedecarboxylase were altered during the design of primer FH0129 to directthe synthesis of the correct amino acids using codons as different aspossible from wt ERG19. A cassette containing the coupled yeast geneswas removed from pFCO1 and inserted into pNGH1-Amp to give pFCO2 inorder to test the ability of the operon to complement the dxs::kan^(r)disruption in FH11 grown on MVA.

Referring to FIG. 2, the cells of plate A were able to grow at 30° C. onLB/Kan medium. The cells of plate B were unable to grow at thenonpermissive temperature of 44° C. on LB/Kan medium, establishing thatE. coli dxs is an essential gene. The cells of plate C were FH11 cellswith R. Capsulatus OFR 2895 contained in pFMH39. These cells were ableto grow at the nonpermissive temperature of 44° C. on LB/Kan/Amp mediumestablishing that R. capsulatus OFR 2895 encodes dxsB. As shown in plateD FH11/pNGH1-Amp cells containing the parent vector of pFMH39 and pFCO2were unable to grow at 44° C. on LB/Kan/Amp medium. The cells of plate Ewere FH11/pFCo2 cells that were able to grow at 44° on LB/Kan/Amp mediumsupplemented with 2 mg/ml mevalonic acid establishing the ability of thesynthetic operon contained within pFCO2 to synthesize IPP. The FH11cells of plate F where able to grow at the nonpermissive temperature of44° C. on LB/Kan medium containing about 0.3 mg/ml methrylerthritol.Thus, CFH11/pFCO2 transformants did not grow at the restrictivetemperature of 44° C. unless MVA (50 mg/L) was added to the growthmedium, establishing the ability of the synthetic operon to directsynthesis of IPP from MVA in E. coli.

1. A transgenic bacterial host cell comprising a genome that comprises:a disruption of a first gene that functionally impairs the first gene,wherein the first gene is an endogenous gene in the methylerythritolphosphate (MEP) pathway; and a transgene under control of an exogenousinducible promoter, wherein said transgene functionally replaces thedisrupted first gene, wherein the transgene is either the same gene asthe disrupted gene from the same organism, or the transgene encodes afunctional equivalent of The disrupted gene from a different organism,wherein the transgenic bacterial host cell further comprises genesencoding phosphomevalonate kinase, mevalonate kinase, and mevalonatediphosphate decarboxylase.
 2. The transgenic bacterial host cell ofclaim 1, wherein the first gene is an endogenous dxs gene.
 3. Atrausgenic bacterial host cell comprising a genome that composes: adisruption of a first gene that functionally impairs the first gene,wherein the first gene is an endogenous gene in the methylerythritolphosphate (MEP) pathway, wherein the first gene is an endogenous dxsgene; a transgene under control of an exogenous inducible promoter,wherein the transgene functionally replaces the disrupted first gene;and genes encoding phosphomevalonate kinase, mevalonate kinase, andmevalonate cliphosphate decarboxylase.
 4. The trausgenic bacterial hostcell of claim 3, wherein the bacterial host cell is selected from thegroup consisting of E. coli and S. typhimurium.
 5. A transgenicbacterial host cell comprising a genome that comprises: a disruption ofa first gene that functionally impairs the first gene, wherein the firstgene is an endogenous gene in the methylerythritol phosphate (MEP)pathway; a disruption of a second gene that functionally impairs thesecond gene, wherein the second gene is an endogenous gene downstreamfrom the first gene in the MEP pathway; and a transgene thatfunctionally replaces the disrupted second gene, wherein the transgeneis either the same gene as the disrupted gene from the same organism, orthe transgene encodes a functional equivalent of the disrupted gene froma different organism, wherein the transgenic host cell farther comprisesgenes encoding phosphomevalonate kinase, mevalonate kinase, andmevalonate diphosphate decarboxylase.
 6. The transgenic bacterial hostcell of claim 5, wherein the first gene is an endogenous dxs gene. 7.The transgenic bacterial host cell of claim 6, wherein the bacterialhost cell is selected from the group consisting of E. coli and S.typhimurium.
 8. The transgenic host cell of claim 5, wherein thetransgene is from a species other than the bacterial host cell.
 9. Thetransgenic host cell of claim 5, wherein the transgene is from the samespecies as the bacterial host cell.
 10. The transgenic host cell ofclaim 5, wherein the transgenic bacterial host cell is selected from thegroup consisting of E. coli and S. typhimurium.
 11. A method ofscreening antibacterial and herbicidal agents for activity against anorganism, the method comprising the steps of: obtaining a transgenicbacterial host cell whose genome comprises a disruption of a first genethat functionally impairs the first gene, wherein the first gene is anendogenous gene in the Methylerythritol phosphate (MEP) pathway, adisruption of a second gene that functionally impairs the second gene,wherein the second gene is an endogenous gene downstream from the firstgene in the MEP pathway, and a transgene that functionally replaces thedisrupted first and/or second gene, wherein the transgene is either thesame gene as the disrupted gene from the same organism, or the transgeneencodes a functional equivalent of the disrupted gene from a differentorganism; providing a test culture and a control culture of thetransgenic bacterial host cell; complementing the disruption of the MEPpathway genes of the transgenic bacterial host cells of the test cultureand the control culture by (a) contacting the transgenic bacterial hostcell with a chemical supplement that bypasses the disrupted first and/orsecond gene in the MEP pathway, and/or (b) expressing the transgene inthe transgenic bacterial host cell that functionally replaces thedisrupted first and/or second gene in the MEP pathway; contacting thetransgenic bacterial host cell of the test culture with a test agent;and comparing growth of the transgenic bacterial host cell of the testculture to the growth of a bacterial host cell of the control culture,wherein the killing of the transgenic bacterial host cell of the testculture and the viability of the transgenic bacterial host cell of thecontrol culture indicates that the test agent is an effectiveantibacterial or herbicidal agent against the organism.
 12. The methodof claim 11, wherein the bacterial host cell is selected from the groupconsisting of E. coli and S. typhimurium.
 13. The method of claim 11,wherein the transgenic host cell further comprises genes encodingphosphomevalonate kinase, mevalonate kinase, and mevalonate diphosphatedecarboxylase, wherein the test culture and control culture of thetransgenic bacterial host cell are optionally contacted with mevalonicacid (MVA) to bypass the MEP pathway.
 14. The method of claim 11,wherein the first gene is an endogenous dxs gene.
 15. The method ofclaim 11, wherein the transgene is from a species other than thebacterial host cell.
 16. The method of claim 11, wherein the transgeneis from the same species as the bacterial host cell.
 17. The method ofclaim 11, wherein the transgenic bacterial host cell is cultured underconditions to express the transgene.
 18. A method of screeningantibacterial and herbicidal agents for activity against an organism,the method comprising the steps of: obtaining a transgenic bacterialhost cell whose genome comprises a disruption of a first gene thatfunctionally impairs the first gene, wherein the first gene is anendogenous gene in the Methylerythritol phosphate (MEP) pathway, and atransgene that functionally replaces the disrupted first gene, whereinthe transgene is either the same gene as the disrupted gene from thesame organism, or the transgene encodes a functional equivalent of thedisrupted gene from a different organism; complementing the disruptionof the first gene of the transgenic bacterial host cells of the testculture and the control culture by (a) contacting the transgenicbacterial host cell with a chemical supplement that bypasses thedisrupted first gene in the MEP pathway, and/ or (b) expressing thetransgene in the transgenic bacterial host cell that functionallyreplaces the disrupted first gene in the MEP pathway; contacting thetransgenic bacterial host cell of the test culture with a test agent;and comparing growth of the bacterial host cell contacted by the testagent to the growth of the bacterial host cell of the control culture,wherein the killing of the transgenic bacterial host cell of the testculture and the viability of the transgenic bacterial host cell of thecontrol culture indicates that the test agent is an effectiveantibacterial or herbicidal agent against the organism.
 19. The methodof claim 18, wherein the first gene is an endogenous dxs gene.
 20. Themethod of claim 18, wherein the test culture of the transgenic bacterialhost cell is cultured under conditions to express the transgene.
 21. Themethod of claim 18, wherein the transgenic host cell further comprisesgenes encoding phosphomevalonate kinase, mevalonate kinase, andmevalonate diphosphate decarboxylase, and wherein the test culture andcontrol culture of the transgenic bacterial host cell are optionallycontacted with mevalonic acid (MVA) to bypass the MEP pathway, whereinwhen an agent renders the transgenic bacterial host cell inviable whenthe host cell is (a) contacted with a chemical supplement that bypassesthe disrupted first gene in the MEP pathway, in the absence of MVAand/or (b) when the transgene in the transgenic bacterial host cell thatbypasses the disrupted first gene in the MEP pathway is expressed in theabsence of MVA, and wherein the transgenic host cell is viable whencontacted with MVA, the agent is an effective antibacterial orherbicidal agent against the organism.
 22. A method of screeningantibacterial and herbicidal agents for activity against an organism,the method comprising the steps of: obtaining a transgenic bacterialhost cell whose genome comprises: (1) a disruption of a first gene thatfunctionally impairs the first gene, wherein the first gene is anendogenous gene in the Methylerythritol phosphate (MEP) pathway, (2) adisruption of a second gene that functionally impairs the second gene,wherein the second gene is an endogenous gene downstream from the firstgene in the MEP pathway, (3) a transgene that functionally replaces thedisrupted first and second genes, wherein the each transgene isindependently either the same gene as the disrupted gene from the sameorganism, or encodes a functional equivalent of the disrupted gene froma different organism, and (4) genes encoding phosphomevalonate kinase,mevalonate kinase, and mevalonate diphosphate decarboxylase; providing afirst and second test culture and a first and second control culture ofthe transgenic bacterial host cell; complementing the disruption of theMEP pathway genes of the transgenic bacterial host cells of the testcultures and the control cultures, wherein the complementation comprisesat least one of the following: (a) contacting the transgenic bacterialhost cell with a chemical supplement that bypasses the disrupted secondgene in the MEP pathway, (b) expressing the transgenes in the transgenicbacterial host cell that functionally replace the disrupted first andsecond genes in the MEP pathway, or (c) contacting the transgenicbacterial host cell with a chemical supplement that bypasses thedisrupted first gene and expressing the transgene in the transgenicbacterial host cell that functionally replaces the second gene in theMEP pathway; bypassing the MEP pathway in the first test culture andfirst control culture by contacting the first test culture and the firstcontrol culture with mevalonic acid; contacting the transgenic bacterialhost cell of the test cultures with a test agent; and comparing growthof the transgenic bacterial host cell of the test cultures to the growthof the transgenic bacterial host cell of the control cultures, whereinwhen first test culture is viable and the second test culture isinviable, the agent is an effective antibacterial or herbicidal agentagainst the organism.